1. Field of the Invention
The present invention relates to continuous sample detection, and in particular, to the use of standards for determining the performance, stability, or reproducibility of the measuring instrument.
2. Discussion of Related Art
The effects of a radioactive tracer introduced into an organism may in part be determined by removing tissue samples for analysis. A known method of detecting these radioactive tracers is to pass extracts, digests, and other solutions derived therefrom through a chromatographic column to separate their constituents into various fractions, in the usual fashion. The eluate from the chromatographic column can then be passed through a device for detecting the radioactivity.
Radioactivity detection is often practiced with a flow-through detector that continuously monitors the radiation from sample flowing through a cell. When the continuous flow to the cell is the eluate from a high performance liquid chromatography column (HPLC) large amounts of information can be obtained through numerical and graphical analysis.
In a known detection technique, the eluate is continuously mixed with a scintillating solution and passed through a transparent tubular cell mounted in a sample block inside a light-tight box. Alternatively, scintillation solution need not be used and instead the eluate is passed over insoluble scintillator particles within the cell. The faint light coming from the tubular cell is detected by a pair of photomultiplier tubes on opposite sides of the cell. See for example, U.S. Pat. No. 4,194,117.
External standardization has been used in liquid scintillation systems to determine the quality of individual samples, especially for quenching phenomena. With that technique, an external radioactive source is brought near the sample to determine how the scintillation process varies in response to known radioactive stimulation. See for example, U.S. Pat. Nos. 3,609,361; 3,188,468; and 3,381,130. For other quenching correction techniques see U.S. Pat. Nos. 4,008,393 and 4,292,520. See also U.S. Pat. Nos. 3,935,449 and 4,967,048.
Calibrating the accuracy of a continuous flow radiochromatography system has problems that are absent from a discontinuous system. Discontinuous systems can work from a plurality of samples and vials that are conveyed to a liquid scintillation detector for discrete measurements. With such a system, sealed standards can be interspersed with the sample vials so that calibration measurements can be made periodically. Systems for measuring the scintillation of discrete vials are shown in U.S. Pat. Nos. 4,634,869; 4,833,326; and 5,146,093.
Known methods for checking performance of a continuous-flow radioactivity monitor involve repetitive measurement of known standard samples followed by statistical data treatment of the measured results. The known methods have disadvantages.
Sealed Standards
The normal counting cell may be removed and replaced with a sealed standard, which is counted under the same conditions, time after time. This sealed standard can be a simple glass tube filled with a mixture of radioactive material and scintillating solution.
By observing the trend of such countings and applying well-known mathematics to the results, the reproducibility of the instrument can be assessed. If the activity level of the standard is known, the counting efficiency of the instrument for the particular conditions employed can be determined.
If several standards of different known activity levels are sequentially counted, it is possible to establish whether or not the system is linear with respect to activity. If several standards, each of a different isotope, are counted, usually with different counting windows (different energy ranges) it is possible to determine the quality of dual-isotope separation and whether or not that changes with time.
Changing from the flow-cell to a sealed standard is difficult. In most instruments, the cover must be removed, the high voltage disabled, plumbing fittings disconnected, the cell removed (potentially causing problems with ambient light as explained below), the standard holder installed, the cover replaced, and the high voltage reestablished. Finally the measurement can be made. Then all of the forgoing steps must be reversed to reestablish normal flow cell operation.
If more than one sealed standard is examined, after each measurement the high voltage must be turned off, the standard holder exchanged, the high voltage turned on again, and then the new standard measured. After the second standard, the steps are repeated and so on, for each standard.
The high voltage must be turned off each time a cell or the standard holder is removed or inserted. Exposure of a photomultiplier to ambient light with the high voltage applied can cause irreparable damage, since the consequential high current flows will likely "strip" the photocathodes.
Regardless, removing the high voltage is inherently problematical, because photomultipliers are most quiet (i.e., least electronic noise) when kept at constant high voltage for long periods. Similarly, even with the high voltage off, exposure to ambient light can cause photocathodes to become light activated, requiring then a lengthy period of dark adaptation to reduce background noise to minimum levels.
Similarly, removal and ambient illumination of cells packed with solid scintillators (particularly the popular yttrium silicate) cause light activation, again requiring a lengthy period of dark adaptation before the cell reaches its lowest backgrounds.
Repetitively disconnecting and reconnecting fittings is an invitation to leaks, especially at the high pressures that are apt to be encountered in HPLC. Cleanup of leakage of radioactive solutions may have heavy consequences.
Filling Cell with Standard Solution
Because replacing a cell with a standard is so unsatisfactory, some methods leave the normal counting cell in place. The cell can be directly filled with a standard solution introduced from a syringe mounted on a Luer fitting through a selector valve (which valve increases the potential for leaks and the generally disadvantageous, system dead volume).
The test starts with the pumping of wash solution through the system to clear residual activity before the cell is filled with a standard solution of radioactivity and scintillator via the syringe. A static test measurement is then made, and the cell is cleared of its standard activity by washing, before the system is returned to service.
This method is only suitable when the counting cell is empty and is being used with liquid scintillator or for Cerenkov counting. It is unsatisfactory for packed cells because fine particles with large total surface areas are normally employed. Back pressures are high, (typically 300-1500 psi.) and uniform filling of such a cell with a syringe cannot be done with confidence. Also, the physical force required, including the reciprocation of a syringe, and the inevitable air displacement, disrupts the packing, introducing voids, repositioning particles, channeling liquid flow, etc.
Occasionally, cells become contaminated. Reasonable washing does not succeed in removing all activity, leading to inaccurate test results. This problem is far more likely with packed cells than with liquid cells.
The overall consumption of standard increases cost, including the currently escalating disposal cost. Moreover, filling a cell via a syringe requires overages to fill the syringe, then to displace the previous wash, and also the fill the lines. Also, daily repetitive use of the same standard solution requires mixing a large amount of standard at one time. This idle supply of radioactivity (of questionable stability) effectively subtracts from the user's legal authority to possess radioactive substances. Linearity checking or the need for different isotopes will multiply the need and these problems.
Also, when a new batch of standard solution is made, its calibration can be affected by the purity of the solvent, varying scintillator purity, the concentration of the scintillator, and the radioactive standard.
Dynamic Measurements
In HPLC systems dynamic (transient) measurements are possible using sampling devices which allow automatic repetitive runs. Standards may be loaded among the samples being measured, but they again impact the costs, waste disposal problems, and other difficulties cited above.
As noted above, checking an instrument using a scintillator-packed cell requires a high pressure pump, which would more than likely be the pump used for chromatography; another pump would mean plumbing rearrangement. Thus to avoid replumbing, one must pass the standard solution through the HPLC column. The measurement will be dynamic and, for each peak, of relatively short duration with the standard peak passing through the cell as it might during a chromatography run.
Solid scintillators are often contaminated and the higher the level of activity, the more likely the contamination. Contaminants in effect provide false high readings, making the test rather pointless unless some form of compensation is effected. The logical compensation would be counting the background and subtracting it, but that is a major source of error unless lengthy counts are performed, which would keep the instrument out of service for excessive periods.
Automatic samplers have their own inherent errors of sample size and including them in a system broadens the potential for overall error. Also, dynamic measurement, unless at quite high activity levels, is not as accurate as a longer static measurement.
Since an HPLC run is usually involved, the accuracy of the measurement is subjected to the vagaries of the HPLC system; the entire process is tested, not just the instrument. For example, the HPLC column normally degrades with use and so too would the measurement.
Also, the total test time with the HPLC column would be excessively long, even though the measurement of the radioactive standard is likely to be inadequate in time. Moreover, to measure a dilution series for linearity requires multiple runs and unacceptable total times. Even if the HPLC column is bypassed in an attempt to accelerate the testing procedure, plumbing changes become necessary with all of the problems previously cited.